Abstract
Voltage-dependent anion channel-1 (VDAC1) is a highly regulated β-barrel membrane protein that mediates transport of ions and metabolites between the mitochondria and cytosol of the cell. VDAC1 co-purifies with cholesterol and is functionally regulated by cholesterol, among other endogenous lipids. Molecular modeling studies based on NMR observations have suggested five cholesterol-binding sites in VDAC1, but direct experimental evidence for these sites is lacking. Here, to determine the sites of cholesterol binding, we photolabeled purified mouse VDAC1 (mVDAC1) with photoactivatable cholesterol analogues and analyzed the photolabeled sites with both top-down mass spectrometry (MS), and bottom-up MS paired with a clickable, stable isotope-labeled tag, FLI-tag. Using cholesterol analogues with a diazirine in either the 7 position of the steroid ring (LKM38) or the aliphatic tail (KK174), we mapped a binding pocket in mVDAC1 localized to Thr83 and Glu73, respectively. When Glu73 was mutated to a glutamine, KK174 no longer photolabeled this residue, but instead labeled the nearby Tyr62 within this same binding pocket. The combination of analytical strategies employed in this work permits detailed molecular mapping of a cholesterol-binding site in a protein, including an orientation of the sterol within the site. Our work raises the interesting possibility that cholesterol-mediated regulation of VDAC1 may be facilitated through a specific binding site at the functionally important Glu73 residue.
Highlights
For LKM38 we identified two paired features consistent with peptide-LKM38 adducts
Irradiating LKM38 prior to mixing with mVDAC1 does not result in significant labeling, in contrast to KK174 which does (Fig. 8)
The fact that multiple cholesterol molecules remain bound to VDAC after detergent solubilization and purification indicates a specific interaction [11, 12], which is likely mediating effects such as the modulation of VDAC gating [6, 11]
Summary
Cholesterol analogues photolabel mVDAC1 with variable efficiency and a stoichiometry of one. To determine cholesterol-binding sites in VDAC1, we photolabeled WT mouse VDAC1 (mVDAC1) with two cholesterol analogues that have a photoactive diazirine in either the 7-position (LKM38) [21] or the aliphatic tail (KK174) (see Fig. 1A for structures and supplemental materials for KK174 synthesis). These reagents were designed to permit mapping of cholesterol binding pockets by placing the diazirine in different positions. The black lines represent b ions that do contain a ligand adduct and were identified by searching the fragmentation data against a database in which the ligand mass was added to the N-terminal methionine.
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